Osteopontin Is Cleaved at Multiple Sites Close to Its Integrin-binding Motifs in Milk and Is a Novel Substrate for Plasmin and Cathepsin D
Osteopontin Is Cleaved at Multiple Sites Close to Its Integrin-binding Motifs in Milk and Is a Novel Substrate for Plasmin and Cathepsin D
复制标题
DOI：
10.1074/jbc.m109.075010
复制
发表时间：
2010-03-12
影响因子：
4.8
通讯作者：
Sorensen, Esben S.
中科院分区：
文献类型：
--
作者：
Christensen, Brian;Schack, Lotte;Sorensen, Esben S.
骨桥蛋白（OPN）是一种高度修饰的整合素结合蛋白，存在于大多数组织和体液中，参与许多生物学过程。OPN功能的重要调节是通过磷酸化和蛋白水解加工介导的。凝血酶和基质金属蛋白酶在整合素结合Arg-Gly-Asp序列附近的蛋白水解切割调节OPN的功能及其整合素结合特性。在这项研究中，7个N-末端OPN片段来源于蛋白水解裂解已被其特征在于从人乳。切割位点的鉴定表明，所有片段均含有Arg-Gly-Asp（145）序列，并通过Leu（151）-Arg（152）、Arg（152）- Ser（153）、Ser（153）-Lys（154）、Lys（154）-Ser（155）、Ser（155）-Lys（156）、Lys（156）-Lys（157）、或Phe（158）-Arg（159）肽键。六个裂解不能归因于凝血酶或基质金属蛋白酶活性，而Arg（152）-Ser（153）处的裂解与凝血酶对OPN的特异性相匹配。乳中的主要蛋白酶纤溶酶水解与凝血酶相同的肽键，但其主要切割位点被鉴定为Lys（154）-Ser（155）。另一种内源性牛奶蛋白酶，组织蛋白酶D，切割Leu（151）-Arg（152）键。还在尿液中鉴定出与纤溶酶活性对应的OPN片段，表明纤溶酶对OPN的切割并不局限于牛奶。纤溶酶，而不是组织蛋白酶D，OPN的裂解增加了α（V）β（3）-或α（5）β（1）-整联蛋白介导的细胞粘附。纤溶酶和凝血酶裂解的OPN介导了类似的细胞粘附，表明纤溶酶可以是OPN活性的有效调节剂。这些数据表明，OPN在其整合素结合基序附近对切割高度敏感，并且该蛋白是纤溶酶和组织蛋白酶D的新型底物。
Osteopontin (OPN) is a highly modified integrin-binding protein present in most tissues and body fluids where it has been implicated in numerous biological processes. A significant regulation of OPN function is mediated through phosphorylation and proteolytic processing. Proteolytic cleavage by thrombin and matrix metalloproteinases close to the integrin-binding Arg-Gly-Asp sequence modulates the function of OPN and its integrin binding properties. In this study, seven N-terminal OPN fragments originating from proteolytic cleavage have been characterized from human milk. Identification of the cleavage sites revealed that all fragments contained the Arg-Gly-Asp(145) sequence and were generated by cleavage of the Leu(151)-Arg(152),Arg(152)- Ser(153), Ser(153)-Lys(154), Lys(154)-Ser(155), Ser(155)-Lys(156), Lys(156)-Lys(157), or Phe(158)-Arg(159) peptide bonds. Six cleavages cannot be ascribed to thrombin or matrix metalloproteinase activity, whereas the cleavage at Arg(152)-Ser(153) matches thrombin specificity for OPN. The principal protease in milk, plasmin, hydrolyzed the same peptide bond as thrombin, but its main cleavage site was identified to be Lys(154)-Ser(155). Another endogenous milk protease, cathepsin D, cleaved the Leu(151)-Arg(152) bond. OPN fragments corresponding to plasmin activity were also identified in urine showing that plasmin cleavage of OPN is not restricted to milk. Plasmin, but not cathepsin D, cleavage of OPN increased cell adhesion mediated by the alpha(V)beta(3)- or alpha(5)beta(1)-integrins. Similar cellular adhesion was mediated by plasmin and thrombin-cleaved OPN showing that plasmin can be a potent regulator of OPN activity. These data show that OPN is highly susceptible to cleavage near its integrin-binding motifs, and the protein is a novel substrate for plasmin and cathepsin D.